Diet composition and selection of Père David's deer in Hubei Shishou Milu National Nature Reserve, China

Abstract Hubei Shishou Milu National Nature Reserve is an ideal place to restore the wild population of Père David's deer (Elaphurus davidianus). Understanding foraging ecology and diet composition is essential for assessing population development or establishing long‐term effective conservation measures for endangered species. However, little is known about the diet composition of Père David's deer and its diet selection mechanism. In this study, we used stable isotope technology to investigate the diet composition of Père David's deer according to various tissues (i.e., fur, muscle, liver, heart, and feces) and seasons, and evaluated the correlation between the nutrient composition of plants and diet composition. Bayesian isotope analysis showed that the autumn and winter diet estimated by fur and fecal samples indicated a diet dominated by C3 grasses (42.7%–57.2%, mean), while the summer diet estimated by muscle and liver samples was dominated by C3 forbs (30.9%–41.6%, mean). The Pearson correlation test indicated that the contribution of winter diet composition reflected by fur and fecal samples was associated with correlations with crude protein (r = .666, p < .01) and soluble sugars (r = .695, p < .01). The results indicated that crude protein and soluble sugars were important factors influencing the winter diet selection of Père David's deer. In the context of the current reintroduction facing many challenges, such as habitat fragmentation, wetland degradation, and human disturbance, comprehensively evaluating the diet selection mechanism of Père David's deer under different resource specificities and temporal changes should be considered in the future.


| INTRODUC TI ON
Père David's deer (Elaphurus davidianus) is one of the national key protected animals belonging to Mammalia, Artiodactyla, Cervidae, and Elaphurus (Cao, 1985). Père David's deer became extinct in the wild in 1900 due to war, poaching, and habitat deterioration. In the 1980s, China began to implement the reintroduction project of Père David's deer and first released captive Père David's deer to nature in 1998 (Ding, 2017). With its reintroduction, ex situ conservation and population restoration have gained a great deal of attention in China Xu & Yu, 2019;Xue et al., 2022).
Understanding diet composition is essential for assessing population development or establishing long-term effective conservation measures for endangered species. However, little is known about the mechanisms of diet selection, food assimilation, and the interrelationships between Père David's deer and their habitat due to the limitations of research methods and the particularity of research objects (Ding, 2009;Hua et al., 2020;Wang & Wang, 2011). Père David's deer mainly lives in swampy and muddy areas and feeds on the young branches and leaves of grasses and some legumes (Ding, 2004). To study the diet composition of Père David's deer more systematically, Ding et al. (1989) (Hua et al., 2020). To further assess the reintroduction of Père David's deer efforts, researchers also need effective information on changes in diet across seasons, including long-term diet composition and factors that mediate dietary variation.
Hubei Shishou Milu National Nature Reserve is the habitat of the largest wild Père David's deer population in China. It is a typical lake wetland located in the middle reaches of the Yangtze River.
However, due to the construction of levees after floods in 1998 and pluvial flooding in the upper reaches of the Yangtze River, the floating aquatic plants were unable to reach the reserve (Li et al., 2012;Zhang et al., 2018). In addition, following the completion of the Three Gorges Dam, the original beach embankment was isolated from natural water exchange between the Tian'E Zhou Oxbow and the Yangtze River (Ding, 2017;Zhang et al., 2018). Human activities, including planting Italian poplar and burning weeds to open up wasteland, accelerated wetland droughts. The affected hygrophytes were gradually replaced by xerophytes and mesophytes, which further accelerated wetland degradation (Li et al., 2016;Zhang et al., 2013;Zhao et al., 2010), soil erosion (Li et al., 2017), and water pollution (O'Hare et al., 2018;Wolka et al., 2018). Wetland degradation may lead to the destruction of wetland ecosystem structure (Davidson et al., 2018), biodiversity reduction (Janne et al., 2021), productivity and wetland function attenuation (Bouma et al., 2014), and other ecological environment deterioration (Fan et al., 2021). Moreover, changes to the vegetation community structure could have a subsequent impact on the largest wild Père David's deer population in China and on the food source of Père David's deer. This further affects the stability of the wetland ecosystem (Hummel et al., 2018;Motta et al., 2020).
Decreases in plant species abundance, diversity, or coverage can affect the foraging choices or habitat use of Père David's deer by changing plant-cervid interactions (Li et al., 2015(Li et al., , 2016. When Père David's deer population abundance increases, it results in a decrease in the diversity and abundance of plants, as seen in other deer populations in Jiangsu, China, Central Japan, and northeastern Illinois (Anderson et al., 2005;Ding, 2017;Iijima et al., 2013). With the increase in trampling frequency of Père David's deer, the soil bulk density increases, while the soil moisture content decreases (Zhou et al., 2010). Meanwhile, available phosphorus, potassium, and other soil components in the surface layers are also continuously reduced (Ding, 2017;Qian et al., 2008;Zhou et al., 2010). This results in lower plant nutrients and reduced biomass, thus preventing the growth of Père David's population within the national reserves. These current and future problems threatening the protection and management of Père David's deer in China could be addressed through the artificial planting of pasture, the implementation of habitat protection and restoration projects, human intervention in population changes (migration in and out) of the population, and other methods.
Understanding the dietary composition and quantifying the nutrient composition of Père David's deer is the basis for this work. This is closely related to the type of forage to be planted, the assessment of emigration sites, and the direction of habitat restoration (Xue et al., 2022;Zhang et al., 2021).
Stable isotope analysis can provide quantitative information on the dietary contribution of various foods consumed by Père David's deer (De Smet et al., 2004). Compared with traditional methods such as behavioral observations and stomach content analyses, stable isotope analysis can provide longer-term measurements (McCue et al., 2020). This can help us to understand variations in Père David's deer diet and provide essential information for conservation.
Depending on which tissues are analyzed, the stable isotope signature would reflect food assimilation over several time scales, from a few days to the lifetime of the animal (Tieszen et al., 1983). Cell turnover in the tissue is an important factor affecting the turnover rate of different tissues in animals. Taking mammals as an example, feces would reflect food consumption of the last days, the liver for approximately the last month, the heart for the last 1-2 months, and the muscle for the last 3-4 months (Bahar et al., 2014;Caut et al., 2009;Phillips et al., 2014;Roth & Hobson, 2000;Sponheimer et al., 2006).
The fur reflects food consumption during fur growth at different time scales (Ayliffe et al., 2004;Cerling et al., 2006;Schwertl et al., 2005), depending on the segment analyzed (Guo et al., 2008;O'Regan et al., 2008;Rogers et al., 2020;Schwertl et al., 2003). We used multiple deer tissues to evaluate the diet of Père David's deer according to different time scales. We also analyzed the nutrient composition of diverse plant species and combined stable isotopes and nutrient compositions to explore the relationship between the diet composition of Père David's deer and the food's nutrient content. Specifically, we aim to address the following scientific aspects of forecasting: (1) Do the stable isotope signatures of Père David's deer vary among tissue types? (2) Does the diet composition of Père David's deer change with the season? (3) Is the diet composition of Père David's deer influenced by the food's nutrient content? 2 | MATERIAL S AND ME THODS
With a total area of 1567 hm 2 , the reserve is adjacent to the Yangtze River to the south with the Tian'E Zhou Oxbow to the east, located at the southern end of the Jianghan Plain (Zou et al., 2013). There are approximately 72 families, 216 genera, and 321 species of wild higher plants in the reserve. The vegetation types in this area can be divided into three groups: meadow type, swamp type, and aquatic plant type. Among them, herbs are the main plant community, and there are few tree species (Zhang, Li, et al., 2019). Père David's deer is the only large mammal in the reserve. lected. In each sample plot, aboveground plant parts with signs of foraging were collected with stainless-steel scissors in sealed bags, which were kept in ice boxes to avoid wilting of plant leaves due to temperature changes. Plants with high cover (more than one-fifth of the sample plot) but no obvious signs of feeding were also collected.
Fresh fecal samples from the areas a and b were collected in January 2021. Père David's deer were spotted and followed by professional staff at a distance, fresh droppings were picked up along the way. One dung mound was regarded as being created by one deer. Five dung mounds were mixed into one fecal sample. A total of nine fresh fecal samples were collected. Soil and plant tissue on the surface of the feces were cleaned with brushes and tweezers and stored in sealed bags. Fecal samples were considered to show the diet in January. Fur samples were collected in April 2019 and 2020. The shedding period of Père David's deer is from March to April and August to September each year (Ding, 2017). The difference in color and size between adult and immature shedding fur masses of Père David's deer is obvious. Fur was collected through professional staff, relying on observation at a distance to ensure that one fur mass was regarded as being created by one deer. To avoid the effects of age, only the fallen fur masses from the adult Père David's deer were collected and kept in sealed bags for further processing. Approximately three to four fur masses were mixed into one fur sample, and a total of four fur samples were collected in areas a and b each year, for a total of eight fur samples collected in 2019 and 2020. The fur divided into three sections (fur tip, fur middle, and fur root) can be approximately considered to show the diet composition from October to November, December to January, and February to March (Ding, 2017;Guo et al., 2008;Schwertl et al., 2003). Muscle, liver, and heart samples were taken from one individual adult male Père David's deer that died accidentally (intraspecific struggle) on August 5, 2019, and were sliced with a sterile scalpel and stored at −80°C after being collected in sealed bags. A total of five muscle samples, five liver samples, and three heart samples were collected from this Père David's deer. Muscle samples are considered F I G U R E 1 Map of the geographical location and regional distribution of the Shishou Milu National Nature Reserve in Hubei Province, China. Areas a and b are the sampling sites for plant and fecal samples.
to show the diet between May and June (Caut et al., 2009;Phillips et al., 2014;Sponheimer et al., 2006). Heart samples are considered to show the diet between June and July (Bahar et al., 2014;Caut et al., 2009;Roth & Hobson, 2000). Liver samples are considered to show the diet in July (Bahar et al., 2014;Caut et al., 2009;Roth & Hobson, 2000;Sponheimer et al., 2006). Research on live animals was performed following the guidelines of the China Wildlife Protection Law, and all research protocols were approved by Hubei Shishou Milu National Nature Reserve.

| Sample pretreatments and lipid removal
Plant samples were processed immediately upon return to the laboratory. Fresh, mature, insect-free plant leaves were cut off with scissors and gently rinsed with distilled water. The cleaned leaves were immediately sent to a freeze dryer for drying (−40°C in a vacuum freezing dryer and 48 h of dehydration; all subsequent freeze-drying settings were the same). The dried leaves were ground into a fine powder using a ball mill and packaged. The fecal samples were washed with distilled water and sent to a freeze dryer for drying.
After being removed, the samples were ground into a fine powder using a ball mill and packaged.
The fur of Père David's deer can be roughly divided into guard fur, underfur, and intermediate fur, with guard fur being the densest and distributed throughout the body. Guard fur is approximately 40-60 mm long, and its growth rate is approximately 6-10 mm/month (Ding, 2017;Perrin & Campbell, 1980). Eight fur samples (guard fur) were picked out with tweezers and straightened, measured, and divided equally into three sections using scissors, for a total of 24 fur subsamples. After they were washed in an ultrasonic bath using a 2:1 chloroform-methanol solution to remove surface contamination and external lipids (Azorit et al., 2012), the fur subsamples were washed with distilled water and then put into an oven for drying .
To further reduce the effect of lipids on stable isotopes, initially processed fur samples and tissue samples from the previous step were subjected to a 2:1 chloroform-methanol solution for further lipid extraction (Ehrich et al., 2011;Rioux et al., 2019). The mixture was shaken and stored at 4°C overnight (18 h; Folch et al., 1957). The supernatant was then removed by centrifugation for 10 min. This procedure was repeated two times (Folch et al., 1957). After three extractions, the samples were dried in an oven. After being dried for 12 h, they were washed with distilled water and then dried in a freeze dryer. After being removed, they were ground into a fine powder using a ball mill and then packaged. All test tissue samples were degreased samples.

| Determination of plant nutrients
To further explore the effect of plant nutrients on the dietary composition of Père David's deer, freeze-dried and ground plants were used for subsequent plant nutrient analysis. Three replicates were set up for each plant species. The mean value (±SD) of three replicates per species was used as the measurement of nutrient content.
Crude fat determination was determined according to the direct extraction method (Zou et al., 1999). Freeze-dried plant samples (500 mg) were dissolved in 10 ml of HCl for 50 min. Then, 10 ml of ethanol was added, and the fat was extracted with 25 ml of petroleum ether, mixed, and shaken, and the supernatant was collected.
The supernatant was dried (105°C for 2 h) and weighed to calculate the crude fat content (Zou et al., 1999).
The C/N and N contents were determined using an elemental analyzer (Flash EA 1112HT; Thermo Fisher Scientific) in the laboratory of the Food Inspection and Quarantine Center, Shenzhen Custom,

China.
The soluble sugar content was determined according to the anthrone-H 2 SO 4 method (Nakamura, 1968). A standard curve was established with glucose standards. Freeze-dried plant samples (250 mg) were extracted by adding 10 ml of distilled water in boiling water for 30 min, and the supernatant was mixed with ethyl anthranilate-concentrated H 2 SO 4 , shaken, and held in a boiling water bath for 1 min. The absorbance was measured at 630 nm by UV spectrophotometry (Nakamura, 1968).
The crude protein content was determined by the Coomassie brilliant blue method (Hayes, 2020). A standard curve was established with bovine serum protein standards. Freeze-dried plant samples (100 mg) were weighed and extracted with distilled water for 2 h at room temperature, the supernatant was mixed with Kaumas Brilliant Blue G250, and the absorbance was measured at 595 nm with UV spectrophotometry (Hayes, 2020).
Condensed tannin (proanthocyanidins) was determined by the acid butanol method (Wei et al., 2014). Freeze-dried plant samples (50 mg) were dissolved in 1 ml of methanol and mixed with 6 ml of 95% butan-1-ol and 5% concentrated HCl in 10 ml test tubes. The tubes were sealed and placed in a water bath at 95°C for 1 h. The color was observed after cooling to room temperature. The alcoholysis under acidic conditions converts the extended units of condensed tannins into colored anthocyanins. The darker the color, the higher the tannin concentration (Wei et al., 2014).

| Stable isotope analyses
After the pretreatments, the carbon and nitrogen stable isotope ratios of the samples were analyzed using an elemental analyzer (Flash where R is the abundance ratio of heavy isotopes to light isotopes in the sample, 13 C/ 12 C and 15 N/ 14 N. R sample is the measured isotope ratio; R standard is the isotope ratio of reference materials. All results are reported relative to atmospheric nitrogen as the standard for δ 15 N and to Cretaceous belemnite (Belemnitella Americana) from the Peedee Formation of South Carolina for δ 13 C. Laboratory standards (glycine and urea) were run every 12 samples to correct any instances of instrument drift. The analytical precision was ±0.1‰ for 13 C and ±0.2‰ for 15 N.

| Mixing models and statistical analyses
For animals with two or more food sources, the proportion of food in animal diets can be determined according to the isotopic mass balance equation: where δ 13 C i and δ 15 N i represent the carbon and nitrogen isotopic compositions of consumers, respectively; δ 13 C j and δ 15 N j represent the carbon and nitrogen isotopic compositions of possible foods, respectively; ΔC and ∆N represent the carbon and nitrogen isotope discrimination values of nutrient grade, respectively; and f ′ ij is the proportion of food in the consumers' food sources (Parnell et al., 2013;Saito et al., 2001).
To estimate the relative contribution of multiple food sources to Père David's deer diet, the Bayesian isotope mixing model SIMMr (stable isotope mixing models) in R (Jackson et al., 2008;Parnell et al., 2010) was used, as it provides advantages over standard, mass balance multisource mixing models (Phillips & Gregg, 2003). SIMMr can integrate sources of variability associated with multiple sources, trophic enrichment factors, and isotopic values, and its outputs represent true probability density functions rather than a range of feasible solutions (Moore & Semmens, 2008;Parnell et al., 2013;Phillips et al., 2014).
As different tissue types metabolize isotopes at different speeds, before applying a mixing model, these systematic differences must be corrected (Phillips et al., 2014). To more accurately reflect the contribution of each food source to Père David's deer, trophic enrichment factors (TEFs) were used to correct for the enrichment of stable isotope signatures between the tissue and diet of Père David's deer. Since TEF values for various tissues of Père David's deer are not available, the TEFs from the currently published literature were used instead. We are aware that incorrect use of TEF can produce erroneous determinations of food sources (Bond & Diamond, 2011).
Estimates are reported with their 95% credible intervals, and the results are displayed as median, 5% and 95% percentile values (Parnell et al., 2013).
One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to test differences in stable isotope values among the potential food sources (C 3 -F, C 3 -G, and C 4 -G) and fur subsamples. Then, a two-way ANOVA was conducted to evaluate the effects of fur subsamples (fur tip, fur middle, and fur root)

| Stable isotope values of potential food sources and samples of Père David's deer
A total of 16 species of plants were collected in this survey, 14 of which were potential food sources for Père David's deer ( Table 1).
Arundo donax and Polygonum perfoliatum (Other) have been studied to confirm that they are not consumed by Père David's deer (Ding, 2017;Zhang, Fu, et al., 2019;Zhang & Yang, 2017). One-way ANOVA showed that the δ 13 C and δ 15 N values of the three types    ), C 3 -G (C 3 grasses), C 4 -G (C 4 grasses), and Other (avoided). The last line of each type is the data after the combination of that type and different letters denote significant differences at p < .05. "+" and "−" indicate the food preference of Père David's deer (

| Diet composition estimates of Père David's deer
The results of the Bayesian mixture model indicated that the C 3 plants (including C 3 -G and C 3 -F) were the main diet component for TA B L E 2 A summary of two-way ANOVA for δ 13 C and δ 15 N values of fur divided into three sections (fur tip, fur middle, and fur root) and sampling time (2019 and 2020) were selected as treatments F I G U R E 2 Stable isotope signatures of carbon and nitrogen from potential food sources (solid points and error bars, corrected for TEF) and raw data from consumers (fur, muscle, liver, heart, and feces of Père David's deer) in scatter plots. The upper right corner indicates the diet window for the sample response.

| Nutrient content of the potential food source
The contents of various nutrients from these 16 representative plants are listed in Table 3. The moisture content of different plants ranged from 70% to 80%. The difference between C. dactylon with the highest crude fat content and P. australis with the lowest crude fat content of grasses was 0.8%. The range of crude fat content of forbs was larger than that of grasses, from 2.5% to 6.6%. The C/N ratios of C. dactylon and P. asiatica were 20.09 and 18.54, respectively, which were higher than those of other plants in the range of 9%-13%. The nitrogen content of C 3 plants was higher than that of F I G U R E 3 Proportional contribution of potential food sources (50%, 75%, and 95% confidence intervals) to the diet of Père David's deer in different years (2019 and 2020) using segmented fur (tip, middle, and root) and estimated using stable isotopic mixing models.

F I G U R E 4
Proportional contribution of potential food sources (50%, 75%, and 95% confidence intervals) to the diet of Père David's deer using different tissue (muscle, liver, heart, and feces) and estimated using stable isotopic mixing models. Only P. lapathifolium and Apium graveolens contained condensed tannins, and the condensed tannins of P. lapathifolium were higher than those of A. graveolens.
The Pearson correlation test was used to analyze the correlation between the nutrient composition of each food source and its proportional contribution to the diet of Père David's deer (Table 4).

| Diet composition of Père David's deer
Research on the feeding habits of ungulates is essential to understanding the interaction between wild animals and the environment. It is also the basis for evaluating population ecology  issues such as habitat quality, environmental tolerance, and feeding strategies of wild animals . We measured diet composition in multiple tissues, and observed differences in diet composition among feces, fur, and other tissues, which will be useful for determining variations in the diet composition of Père David's deer. We observed that the autumn and winter diets estimated by fur samples indicated a diet dominated by C 3 -G (C 3 grasses, 42.7%-57.2%, mean), and the lowest proportional contribution of C 3 -F (C 3 forbs, 22.9%-31.1%, mean) to the diet.
The summer diet estimated by muscle and liver samples showed that the C 3 -F (30.9%-41.6%, mean) was the main plant type consumed by Père David's deer. In the one-way ANOVA and two-way ANOVA of fur subsamples, we found that the results of the effect of the factor sectioning on the stable isotope signature of the fur subsamples were not the same. However, combined with the analysis of the Bayesian mixture model, we believe there is little variation in the composition of the winter diet of Père David's deer.
We speculated that the reason for the lack of significant change is that the diet of the Père David's deer was similar during this time. Alternatively, for Père David's deer, fur may have been integrated into the average diet during the entire fur growth (Rogers et al., 2020).
Comparing tissues that respond to dietary information on different time scales could provide more complex response information on food selection (Rogers et al., 2020). We observed that C 3 -G was consumed much more from November to January (compared with other months in SIMMr with a probability of approximately 0.41).
This is consistent with the results from the fecal samples (collected in January), which demonstrated greater C 3 -G consumption. Careful analysis of the food consumed by Père David's deer revealed a larger range of proportional contributions from G 3 -G and C 3 -F. We speculate that this is related to the fact that the Père David's deer consumed more C 3 plants than C 4 plants. The deviations of δ 15 N values of feces (COV = 0.98) were larger than those of fur (COV < 0.1).
Fecal samples represent the dietary composition over the previous days and present more individual variability. This indicated that the day-to-day diet is variable, but relatively consistent when considered over several months (Meng et al., 2010;Wang & Wang, 2011).
Muscle, heart, and liver samples were all from the same Père David's deer, which affects our prediction of the summer diet composition.
However, considering the little variation in habitats (Zhang, Fu, et al., 2019;Zou et al., 2013) and that Père David's deer are gregarious (Ding, 2017), our results can still provide information on the summer diet composition of Père David's deer. Smaller sample sizes may also affect the analysis of diets (Phillips et al., 2014), but we can still reveal dietary trends throughout the seasons in endangered species by using several body tissues that have different turnover time.
Similar to Wang and Wang (2011), who found that Père David's deer consumed more C 3 -F plants in spring and summer, we also found that C 3 -F became the main food consumed from May to July. This change is the opposite of the autumn and winter months (October to March). We speculate that these changes may be caused by differences in species richness due to seasonal variations in resources (Seto et al., 2015;Taillon et al., 2006). C 3 -F biomass is higher in spring and summer and lower in autumn and winter, while graminoids (C 3 -G and C 4 -G) remain higher in autumn and winter (Zhang, Li, et al., 2019). This change in diet, brought about by changes in biomass, is more evident in the reintroduction of Père David's deer in the Jiangsu wetland in China. In the coastal wetland of Jiangsu Dafeng Milu National Nature Reserve, exotic S. alterniflora has invaded and become the dominant species (Bao & Shi, 2007;Wang et al., 2006;Zhang et al., 2008). Consequently, S. alterniflora gradually became the main food source of Père David's deer, which resulted in a relatively narrow selection of diet composition (Ding, 2009;Ji et al., 2011;Zhang, 2015;Zhao et al., 2010). When the preferred high-quality food resources are limited, ungulates are forced to use low-quality food resources (Gebert & Verheyden-Tixier, 2001;Miranda et al., 2012). This change in feeding behavior is also consistent with other Cervidae (Dumont et al., 2005;Johnson et al., 2001;Zhang et al., 2020).
Our results were different from the diet composition of Père  (Wang & Wang, 2011;Zhang, 2015). Although the change in habitat shows a change in the diet composition of Père David's deer, gramineous plants remain the main food source of Père David's deer (Ding, 2009;Wang & Wang, 2011;Zhang, 2015). We believe this difference in diet is due to differences in habitat types. The geo- Cervus nippon and geographical location also supported this inference (Takatsuki, 2009). C. nippon mainly feed on Sasa nipponica and other graminoids in southern Hokkaido (northern Japan), while they mainly feed on browse and fruits such as Aucuba japonica, Eurya japonica, and acorns of Lithocarpus edulis in southern Japan (Campos-Arceiz & Takatsuki, 2005;Endo et al., 2017;Takatsuki, 2009). We also observed that Père David's deer were mixed feeders, similar to most Cervidae (Meng et al., 2010), despite differences in the diet composition of Père David's deer and other Cervidae (Zhong et al., 2019). For example, Cervus nippon and Cervus elaphus are also typically mixed feeders (Zhong et al., 2019), with trees and shrubs comprising their main food sources (Cui et al., 2007;Gebert & Verheyden-Tixier, 2001;Krojerová-Prokešová et al., 2010). Thus, we suggest that the availability of food items is an important factor influencing diet composition of Père David's deer. In turn, habitat variation affects the availability of food items (Zhong et al., 2019).

| Effects of nutrient content on diet selection of Père David's deer
The knowledge accumulated in the field of nutritional ecology shows with increasing clarity that animal metabolism and diet selection are associated with synergistic and antagonistic assimilation strategies (Felton et al., 2016). We observed that the crude protein and soluble sugars of plants showed a correlation with their proportional contribution to the diet ( Table 4). The results indicated that Père David's deer selectively fed on plants with high protein and soluble sugar contents in autumn and winter. The energy and nutrients of food are absorbed and distributed to various physiologic functions (Boggs, 2009). Protein is one of the most important nutrients for ungulates (Smith, 1978). For most ungulate herbivores, obtaining adequate protein is an important factor affecting diet selection in winter (Berteaux et al., 1998;Demment & Soest, 1985;Illius & Gordon, 1990;Jarman, 1974;Workman & Schmitt, 2012). A study showed that to maintain the metabolic requirements of Odocoileus virginianus in winter, the crude protein content in the diet should be within 13%-16% of the dietary intake (Soest, 2018). In winter, the mean crude protein content of the 14 plant species mainly consumed by Père David's deer in Shishou was 8.36% (Table 3). The crude protein content in the diet of C. elaphus in Europe reaches 5.7% and 5.0% to ensure their protein requirements (Maloiy et al., 1970;Verheyden-Tixier et al., 2008;Yousef Elahi & Rouzbehan, 2008).
This suggests that Père David's deer in Shishou can obtain protein to ensure basic needs in winter. Soluble sugars are also an important energy source for ungulates (Zhang, 2015). Soluble sugars increase rumen fluid volume and dilution rate (Schingoethe et al., 1980;Windschitl & Stern, 1988). It also promotes the absorption and utilization of protein in ruminant (Beever et al., 1978). A study by David's deer are fond of eating P. lapathifolium (Ding, 2017;Zhang, Fu, et al., 2019;Zhang & Yang, 2017). Tannin, as a typical plant secondary metabolite, has attracted extensive attention. Tannins have the ability to bind and precipitate proteins, which affects the protein and nitrogen retention rates of ungulates (Estell, 2010;Qiu, 2016).
Tannins can combine with carbohydrates to a certain extent and affect the nutrient absorption of food by ungulates (Mueller-Harvey, 2006). The intake of tannins by Père David's deer is similar to that by other Cervidae. Bergvall and Balogh (2009) and Bergvall and Leimar (2005) showed Dama dama consumed high-tannin food even in the presence of a low-tannin option. The behavior confirmed that the Cervidae could keep the plant's secondary metabolites at safe levels by adjusting feeding patterns and their bioinvertase system (Chapman et al., 2010;Champagne et al., 2020;Sorensen et al., 2005;Verheyden-Tixier & Duncan, 2000). These results indicate that tannins affect diet selection, but have little effect on Cervidae diets in natural environments (Champagne et al., 2020).

| CON CLUS IONS
Our results showed that the autumn and winter diets estimated by fur and fecal samples indicated a diet dominated by C 3 grasses

ACK N OWLED G M ENTS
We thank Professor Fenxiao Huang for her assistance in plant sorting and identification. We thank Shishou David's Deer National Nature Reserve for supporting our study in the field. We also thank Pengfei Li, Xuemeng Tang, and Jingyi Dai for their assistance in the field and/or laboratory.

CO N FLI C T O F I NTE R E S T
The author declares no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in Dryad at https://doi.org/10.5061/dryad.sqv9s 4n7c.